DIFFERENTIAL EXPRESSION OF INDUCIBLE NO SYNTHASE IN 2 MURINE MACROPHAGE CELL-LINES

Although primary macrophages and most murine macrophage cell lints suc h as RAW 264.7 cells respond to interferon-gamma (IFN-gamma) and/or li popolysaccharide (LPS) by producing large amounts of nitrite, i.e. the oxidation product of nitric oxide (NO) produced by inducible NO synth ase (iNOS), other cell lines like P388.D1 cells do not produce signifi cant amounts, To gain insight into the signalling pathway that leads t o the induction of iNOS activity, we compared iNOS expression in RAW 2 64.7 and P388.D1 cells. We showed that IFN-gamma binds to each cell li ne with a similar affinity. Furthermore, no differences in iNOS eerie structure were detectable by Southern blot analysis. Even though no si gnificant nitrite secretion was found in the supernatant of P388.D1 ce lls stimulated with IFN-gamma and/or LPS. iNOS mRNA expression was ind uced. In addition, IFN-gamma induced the interferon regulatory factor- 1 (IRF-1) gene and activated tile binding of this factor to its target sequence in the iNOS gene. This binding was recently shown to be nece ssary for iNOS expression. However. in P388.D1 cells. we were unable t o detect the corresponding iNOS protein. These results indicate a defi ciency ill P388.D1 cells which appears to be restricted to the signall ing pathway controlling iNOS protein synthesis. This deficiency does n or affect the overall IFN-gamma biological response, but rather a conv ergent post-transcriptional step common to IFN-gamma and LPS.